Usher syndrome (USH) represents the most prevalent form of inherited recessive deafness associated with blindness. In the inner ear, USH proteins constitute the components of stereocilia bundle and mechanoelectrical transduction (MET) machinery. We have previously identified the CIB2 gene encoding Calcium and Integrin-Binding protein 2 (CIB2) as the cause of USH1 and non-syndromic deafness in diverse populations (Patel et al 2015, Riazuddin et al 2012, Seco et al 2016). CIB2 is expressed in the hair cell stereocilia in rodents, but its exact function was unknown (Riazuddin et al 2012). In the previous funding period, we generated a mouse model carrying the human deafness-related Cib2 variant (Cib2F91S knock-in) and characterized it together with a mouse line lacking CIB2 (Cib2tm1a). We found that both these mouse strains are deaf and have no conventional MET responses in the auditory hair cells, despite the presence of tip links that normally gate the MET channels and apparently unchanged localization of mutant CIB2 in the stereocilia of Cib2F91S/F91S mice. We also found that CIB2 binds to the components of the hair cell MET complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 variants. We concluded that CIB2 is essential for the MET function (Giese et al 2017). This novel element of the MET machinery is particularly interesting because it may be responsible for at least some of the multiple well-known effects of Ca2+ on the hair cell mechanotransduction. Here, we will explore the exact role of CIB2 in MET. Towards this end, we have already generated another knock-in mouse strain (Cib2R186W). R186W variant does not affect CIB2 interaction with TMC1/2 but impairs its calcium buffering ability. Cib2R186W mice will be compared with Cib2F91S allele that impairs CIB2 interaction with TMC1/2, leading to the loss of MET. Our studies also established that CIB2 deficiency results in abnormal growth of the transducing shorter row stereocilia in the hair bundle without affecting non-transducing tallest row stereocilia (Giese et al 2017). Thus, CIB2 may represent an important molecule, linking MET channel activity and stereocilia actin core remodeling. The existence of such link was established in our parallel study (Velez-Ortega et al 2017). Here, we will determine how CIB2 controls the height of the transducing stereocilia in the auditory hair cells. Finally, we have also found that CIB2 deficiency results in decreased number of the inner hair cell (IHC) synapses. Therefore, we will investigate the potential mechanisms of CIB2 involvement in the IHC synaptic function. The expected outcomes of this study are to uncover (a) the precise mechanism of deafness associated with CIB2 deficiency and (b) the physiological role of CIB2 protein in hai...